The process of assembly and maturation of murine leukemia virus (MuLV) is poorly understood. Recent studies have demonstrated that high molecular weight precursor proteins undergo proteolytic cleavage to form the major core and envelope proteins of MuLV particles. The proposed work seeks to determine the intracellular location of these intermediates in virus assembly from the time of synthesis through incorporation into mature virus particles. The development, in our laboratory, of a new method of immunoelectron microscopy and the availability of a series of temperature sensitive (ts) mutants of MuLV blocked in particular stages of assembly now enable us to systematically follow the course of assembly events which culminate in the release of mature virus particles. Immunoferritin labelling after thin sectioning for electron microscopy will be used to locate viral antigens in cells containing the MuLV ts mutants. The ferritin label as well as cellular and viral ultrastructure are observed simultaneously in the electron microscope, allowing us to determine the intracellular (and intraviral) location of the virus structural components at each stage of particle assembly. We will also employ high resolution autoradiography to determine the stage at which viral RNA is added to the forming virus particle. These studies will define the temporal sequence of events that occur during the assembly and maturation of MuLV particles.